Mass spectrometry has proven to be useful for identifying and elucidating the structure of complex molecules, and is particularly important in the study of proteins. In particular, the field of proteomics relies to a large extent on various mass spectrometry techniques to examine proteomes, the total set of proteins generated within a particular cell as derived from its genome, and partial proteomes, such as the set of proteins within a specific organelle within a cell.
There is an obvious limitation in applying mass spectrometric techniques in this area, however, in that it is impossible to determine the identity of a large protein molecule solely on the basis of its mass-to-charge ratio taken from a mass spectrum of the protein, and further information is required for identification and to determine molecular structure. To provide supplemental information, large proteins may be broken into smaller constituent peptides by enzymatic digestion prior to mass analysis and/or may be fragmented within a mass spectrometer by collision-induced dissociation (CID) or by electron capture dissociation (ECD); when the digest (the peptides produced by a digestion) or the fragment ions (the constituents produced by physical fragmentation) derived from a single protein are analyzed in a mass spectrometer, the resulting spectrum may provide a peptide mass fingerprint and/or peptide sequence information through which a protein may be identified and characterized.
As noted above, in typical proteomics applications sample proteomes or partial proteomes contain multiple proteins. But when a mixed sample is digested and analyzed without prior separation, the resulting analysis is complicated, since there is no way to tell which peptides are associated with each other as constituents of a specific protein. Thus, high protein sequence coverage is not usually achieved from a digest of a mixed protein sample. This problem can occur even in cases where conventional steps are taken to separate proteins. For example, when gel electrophoresis is performed to separate proteins spatially, the individual protein spots are not always completely distinct and multiple proteins may be included in a removed spot.
Moreover, in applications in which the aim is to analyze different proteins simultaneously, a mixed protein sample is required, with the result that the chromatogram will contain numerous peaks and the mass spectrum will necessarily contain numerous ions representing peptides derived from a number of different proteins, leading to the aforementioned difficulties in identification and characterization.